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  • PAM3 Stimulation

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    Orlando D. Quintanar Haro
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    This document summarizes a procedure for stimulating RAW 264.7 cells with PAM3 and various proteins to analyze TNF-α secretion. The procedure involves: 1) Seeding RAW 264.7 cells and refreshing media on days 1-2. 2) On day 3, washing cells and adding PAM3 or test proteins at defined concentrations. 3) Incubating cells for 5 hours then taking supernatants to analyze TNF-α secretion using ELISA. Protein concentrations were 0.06 or 0.1 μg/mL depending on dilution used.

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    This document summarizes a procedure for stimulating RAW 264.7 cells with PAM3 and various proteins to analyze TNF-α secretion. The procedure involves: 1) Seeding RAW 264.7 cells and refreshing media on days 1-2. 2) On day 3, washing cells and adding PAM3 or test proteins at defined concentrations. 3) Incubating cells for 5 hours then taking supernatants to analyze TNF-α secretion using ELISA. Protein concentrations were 0.06 or 0.1 μg/mL depending on dilution used.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Scarica in formato DOC, PDF, TXT o leggi online su Scribd
    Segnala contenuti inappropriati
    26 visualizzazioni1 pagina

    PAM3 Stimulation

    Caricato da

    Orlando D. Quintanar Haro
    This document summarizes a procedure for stimulating RAW 264.7 cells with PAM3 and various proteins to analyze TNF-α secretion. The procedure involves: 1) Seeding RAW 264.7 cells and refreshing media on days 1-2. 2) On day 3, washing cells and adding PAM3 or test proteins at defined concentrations. 3) Incubating cells for 5 hours then taking supernatants to analyze TNF-α secretion using ELISA. Protein concentrations were 0.06 or 0.1 μg/mL depending on dilution used.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Scarica in formato DOC, PDF, TXT o leggi online su Scribd
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    di 1

    PAM3 STIMULATION

    Procedure: Day1: Seed RAW 264.7 cells on 6 well plates at 2x106/ well in 2ml DMEM+/+. Day2: Refresh DMEM+/+ Day3: Wash cells 3x in 2ml PBS (37C) and add 2ml DMEM-/a) PAM3 1) Prepare a working solution: 1.1) 150L (30 portions) working solution (132L DMEM -/-, 18L PAM3 [Stock: 1L/L]) 1.2) Add 5L to each wall b) Proteins (TcpF, TcpC-TIR, Azurin and GFP). a. The working concentration of all the proteins is 0.06g/L. 10 L portions should be put in the samples giving 0.6g on 600L (Final concentration of 1g/mL) b. Make a second working solution by diluting the first solution 1:10 (5+45L). 10L portionscorrespond to 0.1g/mL 5L portions correspond to 0.05g/ml c) Incubation for 5 hours d) Take two alicuots of 100L of the supernatant to analyse mTNF- secretion. (R&D ELISA) e) Keep samples on ice or stire at -20C for long time storage.

    Optimal dilution factor for previous experiments was 1/64. Follow R&D manual instruction carefully!

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